2.5 | Ultrasound-induced cytosolic delivery of Saporin
Finally, we demonstrated a control over cells in an ultrasound-induced manner using the current cytosolic protein delivery system. Saporin (Sap) was used as a cargo protein. Sap is a plant-derived ribosome-inactivating protein that leads to cell death through its introduction into the cytosol[35,36]. Sap has no free thiol group; therefore, the amine group of the lysine residues was thiolated with 2-iminothiolane hydrochloride[37,38]. Thiolated Sap was conjugated with PCNDs via disulfide bond formation in the same manner as that used for β-Gal. After intracellular accumulation of PCNDs carrying Sap, the cells were exposed to ultrasound, followed by incubation for 2 days. Alive cells were stained with calcein-AM, and all cells were subsequently analyzed for the presence of green fluorescence of calcein using flow cytometry. The cell viability drastically decreased to below 30% after the treatment with both Sap-carrying PCNDs and ultrasound, while the treatment with Sap-carrying PCNDs without exposure to ultrasound barely reduced the cell viability (Figure 4). Although the cell viability decreased to 66% even in the control experiment wherein PCNDs without Sap were used, there was a significant difference in the cell viability between PCNDs with and without Sap after an exposure to ultrasound. These results indicated that Sap was released into the cytosol upon exposure to ultrasound, resulting in a significant contribution to decreased cell viability.